Serotype distribution, trend of multidrug resistance and prevalence of β-lactamase resistance genes in human Salmonella isolates from clinical specimens in Guizhou, China

Salmonella, one of the major causes of foodborne infections, can cause bacterial foodborne illness. We investigated the serotype distribution, multidrug resistance (MDR), and β-lactamase resistance genes of human Salmonella isolates collected from clinical specimens in Guizhou, China, between 2013 and 2018. A total of 363 Salmonella isolates were collected from clinical specimens at 17 surveillance hospitals. Twenty-four serotypes were identified by sliding agglutination test. S. Enteritidis (33.9%), Salmonella 4,[5],12:i:- (24.0%), S. Typhimurium (16.3%), S. London (6.3%), and S. Derby (3.9%) were the top five serotypes. In 2018, the most common serotype changed from S. Enteritidis to S. Typhimurium. Among the 363 Salmonella isolates, 97.5% of isolates were resistant to at least one class of antimicrobial agents. For cephalosporins, ceftriaxone had the highest resistance rate of 10.5%, and cefepime and cefoxitin were 8.0% and 2.2%, respectively. Three hundred and one (82.9%) Salmonella isolates showed MDR. Salmonella 4,[5],12:i:- had the highest MDR rate with 94.2%, followed by S. London (91.3%) and S. Typhimurium (88.1%). Multidrug resistance rates of Salmonella isolates in Guizhou from 2013 to 2017 increased from 75.8% to 86.7%. Sixteen isolates (4.4%) showed extensive drug resistance. One hundred thirty-four antimicrobial resistance patterns were found. Two hundred and forty-one (66.4%) isolates carried at least one β-lactamase resistance gene. The blaTEM gene (61.2%) was the most prevalent resistant gene in all Salmonella isolates, followed by the blaCTX-M gene (6.1%) and blaOXA-1 gene (4.1%). Our findings showed that the MDR rate of Salmonella isolates from Guizhou province increased year by year. Therefore, systematic and long-term surveillance on MDR Salmonella isolates from clinical patients should be further strengthened.

Introduction Salmonella is one of the most common pathogens of infectious diarrhea worldwide [1]. It is an essential foodborne and zoonotic pathogen. The data from the United States on nontyphoidal Salmonella infections indicated that there were approximately 1.2 million illnesses, 23,000 hospitalizations, and 450 deaths each year [2]. In the European Union, the incidence of Salmonella infections ranked well behind Campylobacter in 2014 [3]. In China, 9.87 million cases of gastroenteritis caused by Salmonella each year [4], and outbreak events due to Salmonella infection sometimes occurred with the most significant number of patients [5]. The data on bacterial infectious diarrhea disease from the National Infectious Disease Report Management System of China ranked Salmonella first in incidence, resulting in 422,325 cases in 2005-2019 [6]. Salmonella enterica serovar Enteritidis (S. Enteritidis) and S. Typhimurium were the first and second serotypes, with 4,140 (36.36%) and 1,502 (13.19%) reported cases, respectively [6]. At the same time, the antimicrobial resistance of Salmonella is also one of the most critical public health problems worldwide. Multidrug-resistant (MDR) Salmonella poses a severe threat to humans. The MDR of Salmonella isolates in the United States was 10.3% from 2004 to 2016 [2]. Salmonella isolates from the ten EU member states were tested for nine classes of antimicrobial agents with an overall high MDR(26%) [3]. The level of antibiotic resistance in Salmonella varies from country to country and is influenced by antibiotic use practices in humans and animals. Salmonella serovars and antimicrobial resistance can display distinct geographic characteristics [7]. From 2011 to 2016, 486 outbreaks of foodborne diseases were reported by various cities and prefectures in Guizhou province through the Foodborne Disease Outbreak Monitoring System. Among the events with clear etiology, Salmonella was the primary pathogen causing bacterial foodborne outbreaks and the largest number of cases in Guizhou province [8]. The surveillance results of foodborne diseases in Guizhou province from 2015 to 2017 showed that Salmonella ranked first among all pathogens, suggesting that Salmonella should be the focus of foodborne disease surveillance in Guizhou province in the future [9]. S. Enteritidis was the dominant serotype in infectious diarrheal cases in Guiyang city of Guizhou Province [10]. Furthermore, previous food surveillance in Guizhou province showed that S. Typhimurium was the most common serotype [11]. To fully interpret the antimicrobial resistance data, it is necessary to describe the underlying antimicrobial-resistant mechanisms of bacteria. However, previous studies did not provide sufficient information on the systematic serotype distribution, the trend of MDR, and the prevalence of β-lactamase resistance gene in clinical Salmonella isolates in Guizhou province. Few data on the trends of historical antimicrobial resistance in Salmonella isolates are available.
This study investigated the serotype distribution, the trend of MDR, and the prevalence of β-lactamase resistance genes of Salmonella isolates from clinical specimens in Guizhou, with a special focus on the consistency between the presence of the β-lactamase resistance gene and β-lactamase antimicrobial phenotypes. These results could provide a reference basis for the scientific prevention and control of Salmonella infection and the rational use of antimicrobials.

Ethics statement
This study was a retrospective study of archived samples. We ensured that all data were fully anonymized. The present study was reviewed and approved by the Ethics Review Committee of Guizhou Provincial Center for Disease Control and Prevention.

Isolates collection and identification
All the cities, including six cities and three autonomous prefectures in Guizhou province of China, were included in this program. Patients with the following symptoms were selected for the study: Three or more episode of diarrhea within 24 hours, watery or sticky stools, mucus or pus-bloody stools. Also, the following criteria were considered for suspicious cases of non-typhoidal Salmonella infection: fever (temperature >38˚C), or with headache, chills, fatigue; nausea, vomiting or abdominal pain. Stool samples from patients with clinical diarrhea were collected to isolate Salmonella from 2013 to 2018. All fecal samples were cultured overnight at 37˚C in local hospitals using MacConkey agar plates (Huankai, Guangdong, China). Systematic biochemical methods were used to identify suspected colonies [12]. All suspicious Salmonella isolates were submitted to the laboratory of Guizhou Provincial Center for Disease Control and Prevention (Guizhou CDC) for further validation and serotyping. In the laboratory, these isolates were recovered by inoculating on nutrient agar plates (Huan Kai, Guangdong, China) and further identified by the API20E identification kit (Biomerieux, France). Identified Salmonella isolates were serotyped by O and H antigen slide agglutination tests (SSI, Denmark) according to the White-Kauffmann-Le Minor scheme [13].

Detection of β-lactamase resistance genes
All Salmonella isolates were screened for seven β-lactamase genes (bla TEM , bla SHV , bla OXA-1 , bla OXA-2 , bla PSE , bla CMY , and bla CTX-M ) using a simplex PCR assay. The primers used to amplify the antimicrobial resistance genes (ARGs) in this study were listed in S1 Table. The PCR assay was performed according to the reported reaction conditions [15,16]. The isolates positive for bla TEM and bla CTX-M genes were further sent for sequencing. The DNA sequences obtained were further aligned using BLAST analysis to identify the subtypes of resistance genes (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
analyze the concordance between the presence of the β-lactamase an and β-lactamase antimicrobial phenotypes. A P value of <0.05 was define as statistical significance.

Detection of β-lactamase antimicrobial resistance genes
PCR results indicated that 241 Salmonella isolates (66.4%) carried at least one β-lactamase resistance gene. The most prevalent was the bla TEM gene (61.2%, 222/363), followed by the bla CTX-M gene (6.1%, 22/363) and bla OXA-1 gene (4.1%, 15/363) ( Table 4). Ten (2.8%) isolates produced both bla TEM and bla CTX-M genes, and one isolates carried bla TEM , bla CTX-M , and bla OXA-1 genes simultaneously. The bla SHV , bla OXA-2 , and bla PER genes were not detected in the Salmonella isolates. According to sequence BLAST online, the 222 Salmonella isolates with the bla TEM gene belonged to bla TEM-1 . Seven subtypes of the bla CTX-M gene were obtained    . The two isolates carrying the bla CMY-2 gene were resistant to more than ten classes of antibiotics, including β-lactamase antibiotics, such as ceftriaxone, cefepime, ampicillin, amoxicillin/clavulanate. Salmonella isolates with different serotypes differed in carrying the β-lactamase resistance gene. Bla TEM  The prevalence of β-lactamase resistance genes of Salmonella isolates varied among regions. The detection rate of bla TEM gene in each city (prefecture) ranged from 44.4% to 81.5%, with the highest detection rate in Bijie. Bla OXA-1 gene was detected in five cities (prefectures), including Bijie, Liupanshui, Guiyang, Tongren and Zunyi, with the highest detection rate in Tongren (9.3%). Bla CTX-M -positive strains were distributed in six cities (prefectures), including Anshun, Guiyang, Qiandongnan, Qianxinan, Tongren and Zunyi, with the highest detection rate in Bijie (9.1%). The bla CMY gene was only detected in Salmonella strains in Tongren.

The concordance between the β-lactamase ARGs and phenotypic antimicrobial resistant
In this study, we compared phenotypic and genotypic resistance to five β-lactamase antimicrobials. There was an overall concordance of 80.4% between phenotypic and genotypic resistance against 363 Salmonella isolates. Of these 279 phenotype-positive Salmonella isolates, 227 (81.3%) contained the β-lactamase resistance genes. However, of 84 phenotype-negative Salmonella isolates, 19 (22.6%) contained the β-lactamase resistance genes. A certain correlation was found between the bla TEM gene and ampicillin (Kappa = 0.452, P < 0.001). Furthermore, the bla CTX-M gene was in good agreement with ceftriaxone and cefepime (Kappa = 0.688, Kappa = 0.655, P < 0.001), as shown in Table 5.

Discussion
Serological identification in this study showed that the number of Salmonella isolates in the top ten serotypes accounted for more than 90%, among which S. Enteritidis, Salmonella 4, [5],12: i:-and S. Typhimurium were the main serotypes. The prevalence of serotypes was similar to those reported in the United States [17], the European Union [18], and Guangdong Province [19]. However, the serotypes were different from the dominant serotype of S. Agona reported in Maanshan city, Anhui province [20]. This information indicated that the distribution of Salmonella serotypes had regional characteristics. S. Enteritidis is mainly associated with contaminated eggs, while Salmonella 4, [5],12:i:-and S. Typhimurium are mainly associated with contaminated pork and pork products. These contaminated foods are easily transmitted to humans through the food chain and cause illness. Future surveillance in Guizhou province should be conducted to determine the source of Salmonella infections and risk factors for disease control and prevention. In this study, Salmonella serotypes became more and more diverse from 2013 to 2018. Meanwhile, 17 isolates could not be classified by serotype, and either new serotypes or other rare serotypes require further investigation. The continued emergence of rare serotypes of Salmonella should be of great concern, as these Salmonella serotypes have the potential to spread and cause public health problems. These results indicate that the dynamic monitoring of Salmonella serotypes and focusing on serotype changes in different regions are significant for targeting the prevention and control of salmonellosis in Guizhou.
Increasing antimicrobial resistance was found in this study, especially to the traditional agents, such as sulfamethoxazole, streptomycin, ampicillin, tetracycline, doxycycline, and nalidixic acid, which was similar to the results reported in some regions of China [21,22], but greatly higher than those of human Salmonella in the European Union [23]. Decreased susceptibility to ciprofloxacin is a current trend in non-typhoidal Salmonella isolates worldwide [24]. In this study, decreased susceptibility to ciprofloxacin was as high as 65.0% (MIC�0.12μg/ mL), which may affect clinical treatment and may lead to treatment failure [24]. Therefore, ciprofloxacin sensitivity should be carefully considered when selecting this antimicrobial agent. MDR Salmonella has become a significant threat to human health. The overall rate of MDR (82.9%) was much higher than that reported in Zhejiang and Anhui, China [25,26]. MDR Salmonella increased from 2013 to 2017 in this study, possibly as a result of antimicrobial misuse in humans. In addition, MDR was widely distributed in various serotypes of Salmonella in this study, especially in Salmonella 4, [5],12: i:-(94.2%) and S. London (91.3%), which were much higher than the results reported in other cities [27][28][29]. In addition, 5.3% of the isolates showed XDR and 16 isolates were resistant to ten or more antibiotics simultaneously. In terms of regional distribution, Tongren city had the highest MDR rate, followed by Liupanshui city. Meanwhile, we discovered that the AMR patterns of Salmonella isolates became more diverse and broader year by year. Our results highlight the serious issue of

PLOS ONE
Salmonella MDR in clinical samples in Guizhou, which may lead to the evolution of Salmonella into a super bacterium and pose a risk to public health [30,31]. Therefore, the enhanced and continuous surveillance of antimicrobial resistance Salmonella in different serotypes and regions should be carried out to further monitor MDR Salmonella with public health concerns. Appropriate and effective antimicrobial agents for the treatment of Salmonella infection should be selected according to drug sensitivity testing in each region. Moreover, a total of four β-lactamase resistance genes were detected in this study, among which bla TEM had the highest detection rate (61.2%), which was similar to the results of human Salmonella studies in Zhejiang [32] and Beijing [33], but different from bla OXA-1 prevalent in Henan [34] and bla CTX-M prevalent in Shanghai [35]. This result may be related to the different types of antibiotics used and the prevalence of serotypes in different regions. It is noteworthy that 130 Salmonella isolates from nine regional pig farms in Guizhou province were detected for β-lactamase resistance genes, of which bla TEM with a detection rate of 85% was consistent with the results of this study [36]. Pork and pork production may be the source of β-lactamase resistance genes in Guizhou province. All the bla TEM -positive isolates in this study were harbored bla TEM-1 gene, which was different from bla TEM-52 , bla TEM-3 , and bla  genes prevalent in other countries [37][38][39]. In addition, we noticed that bla TEM-1 isolates were widely distributed among different serotypes, suggesting that this genotype had no serotype specificity and might be continuously spreading in Salmonella isolates from Guizhou province. Therefore, we should further strengthen the β-lactamase resistance gene surveillance of Salmonella isolates from clinical samples and pork production.
In this study, 6.1% of isolates carried the bla CTX-M gene, which was lower than the detection rate of bla CTX-M reported in Zhejiang [32]. Bla CTX-M gene subtypes were diverse, and the most common subtype was bla CTX-M-55 , which was consistent with other reports such as Sichuan [40] and Guangdong [41]. The above two provinces are closer to Guizhou province, and there may be the cross-regional transmission. However, bla CTX-M- 14 and bla CTX-M- 15 were prevalent in Jiangxi [42] and Beijing [33]. The distribution of bla CTX-M showed significant regional differences. Meanwhile, we found that bla CTX-M was mainly distributed in a few serotypes, especially Salmonella 4, [5],12: i:-, in which more isolates carried the bla CTX-M gene with diverse subtypes. This is consistent with the higher resistant rate of Salmonella 4, [5],12:i:-to cephalosporins than other serotypes in this study.
Bacterial production of the AmpC enzyme is another major resistance mechanism of Gram-negative bacilli to β-lactam antibiotics following ESBLs in recent years [43]. Newly discovered plasmid-mediated AmpC enzymes have gradually increased, and Bla CMY -producing Salmonella has continued to spread in North America, becoming a major concern for cephalosporin resistance in this region [37]. Ceftriaxone-resistant Salmonella isolates in the United States were mainly mediated by the plasmid-encoded bla CMY gene [44]. Plasmid-encoded bla CMY-2 was the most common and destructive β-lactamase, which can seriously affect the therapeutic effect of broad-spectrum cephalosporins [34]. In this study, two isolates with the bla CMY gene were detected and identified as bla CMY-2 by sequencing comparison, which was reported for the first time in Guizhou. The two bla CMY-2 -positive clinical Salmonella isolates were resistant to ceftriaxone, cefepime, ciprofloxacin, azithromycin, and other clinically significant antibiotics. We noticed that isolates carrying bla CMY-2 gene were usually multidrug resistant, which puts tremendous pressure on the prevention and control of multidrug resistant bacteria [45,46]. Therefore, it is essential to strengthen the surveillance of bla CTX-M and bla CMY-2 positive Salmonella isolates and to explore the mechanisms of resistance.
We noticed that the bla TEM gene primarily mediated ampicillin resistance, while the bla CTX-M gene mainly mediated ceftriaxone and cefepime resistance. The results were similar to those reported [33,45], indicating that the antimicrobial resistance of Salmonella was closely related to the presence of antimicrobial resistance genes. However, the 69 ampicillin-resistant Salmonella isolates in this study did not carry the bla TEM gene. The 16 Salmonella isolates resistant to ceftriaxone and 13 Salmonella isolates resistant to cefepime did not carry the bla CTX-M gene, indicating that some of the isolates were not consistent between antimicrobial resistance phenotypes and genotypes. This could be other resistance mechanisms or antimicrobial resistance genes mediating the resistance. Therefore, the mechanism of antimicrobial resistance needs to be studied more comprehensively.
In summary, our study provides the first systematic overview of the serotype distribution, MDR trend, and the prevalence of β-lactamase resistance genes in human Salmonella isolates from clinical specimens between 2013 and 2018 in Guizhou province of China. The distribution of Salmonella serotypes showed regional characteristics. The MDR of Salmonella isolates was at a high level and gradually increased from 2013 to 2017. The ARGs of the isolates showed that the bla TEM gene was the most prevalent, followed by the bla CTX-M and bla OXA-1 genes. It is important to strengthen this platform for ongoing and enhanced surveillance, which is critical in rapidly increasing antimicrobial resistance.
Supporting information S1